Different LED Light Wavelengths and Photosynthetic Photon Flux Density Effect on Colletotrichum acutatum Growth

The study aimed to evaluate the effect of different photon flux density (PFD) and light-emitting diodes (LED) wavelengths on strawberry Colletotrichum acutatum growth characteristics. The C. acutatum growth characteristics under the blue 450 nm (B), green 530 nm (G), red 660 nm (R), far-red 735 nm (FR), and white 5700 K (W) LEDs at PFD 50, 100 and 200 μmol m−2 s−1 were evaluated. The effect on C. acutatum mycelial growth evaluated by daily measuring until five days after inoculation (DAI). The presence of conidia and size (width and length) evaluated after 5 DAI. The results showed that the highest inhibition of fungus growth was achieved after 1 DAI under B and G at 50 μmol m−2 s−1 PFD. Additionally, after 1–4 DAI under B at 200 μmol m−2 s−1 PFD. The lowest conidia width was under FR at 50 μmol m−2 s−1 PFD and length under FR at 100 μmol m−2 s−1 PFD. Various LED light wavelengths influenced differences in C. acutatum colonies color. In conclusion, different photosynthetic photon flux densities and wavelengths influence C. acutatum growth characteristics. The changes in C. acutatum morphological and phenotypical characteristics could be related to its ability to spread and infect plant tissues. This study’s findings could potentially help to manage C. acutatum by LEDs in controlled environment conditions.


Introduction
Nowadays, one of the serious problems is food production contamination by various sources as chemical pesticides, phytotoxins, chemicals for food processing, and others [1]. In food production, the residues of chemical pesticides are present as contaminants in higher than safe concentrations [2][3][4]. However, traditionally, fungicides are used for disease control by routine application [5,6]. Besides this, chemical pesticides negatively affect beneficial organisms in the environment, pollute water, food, soil, and affect animal and human health due to frequent use [2,3,[7][8][9]. Furthermore, the growing demand for safe food leads to alternative plant protection because chemical pesticides have side effects and induce resistance to pathogens [10,11]. Additionally, the European Union (EU) directive 2009/128/EC and Green Deal promotes sustainable pesticides use. Therefore, various physical, biological, and integrated plant protection strategies are applied to prolong and maintain the shelf-life of horticultural crops [12][13][14][15]. Furthermore, plant protection should be based on new substances and technologies for integrated harmful organism control with the least harmful methods for humans and the environment [3,9,12,14,15].
The Colletotrichum genus pathogens complex is one of the primary disease-causing strawberry fruit contaminations. Strawberry anthracnose (Colletotrichum spp.) can infect up to 80% of nurseries plants and reduce yield by over 50%. The Colletotrichum spp. (C. acutatum, C. fragariae, and C. gloeosporioides) infects a wide diversity of hosts and is an important horticultural crop in southern countries. The optimal temperature for pathogen 2 of 12 development is between +26.7 and 32 • C, but it adapts to cooler climatic conditions concerning warm day periods regarding climate change [12,[16][17][18][19][20]. The Colletotrichum species complex comprises about 190 species, of which three species, C. acutatum, C. gloeosporioides, and C. fragariae, cause strawberry anthracnose [19,20]. The microbial contamination of fruits reduces their shelf-life and occurs during harvesting and postharvest [21].
Light is essential for all crops, as it induces photosynthesis, which is crucial for plant growth and development. In addition, in greenhouses and closed environmental conditions, supplemental light eliminates cropping season and improves nutritional quality, decreases nitrate concentration, increases yield quality, and influences plants' grown parameters. The role of light on various crops was evaluated [22][23][24][25]. However, fungal pathogens also react to solar radiation, and it depends on the duration of exposure, wavelengths, and irradiance [26]. Fungi sense light using 11 photoreceptors, which control morphological and physiological responses [27]. Sunlight induces photomorphogenesis, phototropism and serves for signal orientation [28]. The ultraviolet B radiation (UV-B) could positively or negatively affect fungal growth and conidia germination [26]. Fungal responses to light are reflected by conidia production, stress tolerance, pigmentation, virulence, second metabolism, and germination speed [29]. Therefore, LED light use for plant pathogens control relies on sustainable technologies for integrated harmful organism control [13][14][15]. It has been reported that blue LED light could suppress the sporulation and germination of Penicillium spp., Phomopsis spp., Botrytis spp., and Aspergillus spp. Additionally, blue, red, and far-red inhibit Aspergillus spp. and other pathogens [8,9,30,31]. Additionally, blue, green, and red LED lights can induce systemic resistance to pathogens [14]. However, the light exposure time should be economically efficient [32]. In addition, 50-150 µmol m −2 s −1 PFD of blue LED induced B. cinerea resistance in tomatoes [8].
It was previously reported that LED light affects pathogens. However, a lack of different photosynthetic photon flux densities and different wavelengths impact C. acutatum.
In addition, C. acutatum morphological and phenotypical characteristics could be related to its ability to spread and infect plant tissues. Therefore, we assume that different photon flux densities and wavelengths could suppress or stimulate plant pathogens. Furthermore, not all studies evaluated morphological and phenotypical characteristics of C. acutatum that could influence pathogen ability to infect plant tissues and spread. Therefore, the present study aimed to evaluate the effect of different photon flux densities and LED light wavelengths on strawberry C. acutatum growth characteristics in vitro.

Results
The C. acutatum was exposed under different LEDs wavelengths, and PFD revealed morphological and phenotypic characteristics difference. In order to evaluate the intensity of LED light effect on C. acutatum growth characteristics, the isolates exposed B, G, R, FR, and W and 50, 100 and 200 µmol m −2 s −1 PFD. We observed that C. acutatum acted differently under different PFD and LEDs. In addition, the mycelium growth was different for each PFD. The LEDs influenced C. acutatum mycelium growth, acting differently under different light conditions. The lowest C. acutatum mycelium growth at 50 µmol m −2 s −1 PFD observed after 1 DAI was under B and G, at 2 DAI-G, at 3 DAI-FR, and 4 DAI-G ( Figure 1A). In addition, the highest mycelium growth rate was under R at 1-4 DAI, besides at 1 and 4 DAI under FR. The MGI inhibition of C. acutatum mycelium growth under different wavelengths and 50 µmol m −2 s −1 PFD, at 2 DPI reached-0.95% G and 4 DPI 1.45%.
The results showed that at 100 µmol m −2 s −1 PFD, the highest inhibition of C. acutatum mycelium growth was under R after 1 and 3-4 DAI ( Figure 1B). Additionally, the after 2-3 DAI FR compared with other treatments. However, G increased the mycelium growth at 1-4 DAI and B at 1 and 4 DAI. The MGI inhibition at 2 DAI reached 2.38% under FR and at 4 DAI-2.66% under R.
However, the highest inhibition of C. acutatum mycelium growth was observed at 200 µmol m −2 s −1 PFD under B after 1-4 DAI ( Figure 1C). Besides, the highest mycelium growth was observed under FR at 1-3 DAI and R at 2 and 4 DAI. The MGI inhibition rate at 2 DAI-12.33% and 4 DAI-4.69% under B.
Different wavelengths influenced the mycelial growth of the C. acutatum at various PFD. Overall, our data show that the highest inhibition of C. acutatum after 4 DAI was at 50 µmol m −2 s −1 PFD under G, at 100 µmol m −2 s −1 PFD-R, and 200 µmol m −2 s −1 PFD under B.
The results showed that at 100 μ mol m −2 s −1 PFD, the highest inhibition of C. acutatum mycelium growth was under R after 1 and 3-4 DAI ( Figure 1B). Additionally, the after 2-3 DAI FR compared with other treatments. However, G increased the mycelium growth at 1-4 DAI and B at 1 and 4 DAI. The MGI inhibition at 2 DAI reached 2.38% under FR and at 4 DAI-2.66% under R.
However, the highest inhibition of C. acutatum mycelium growth was observed at 200 μ mol m −2 s −1 PFD under B after 1-4 DAI ( Figure 1C). Besides, the highest mycelium growth was observed under FR at 1-3 DAI and R at 2 and 4 DAI. The MGI inhibition rate at 2 DAI-12.33% and 4 DAI-4.69% under B.
Different wavelengths influenced the mycelial growth of the C. acutatum at various PFD. Overall, our data show that the highest inhibition of C. acutatum after 4 DAI was at 50 μ mol m −2 s −1 PFD under G, at 100 μ mol m −2 s −1 PFD-R, and 200 μ mol m −2 s −1 PFD under B.  Figure 2C). The PCA biplots generally showed distinct effects of B and G to R and FR regardless of PFD. The PCA factor loadings, scores, and eigenvalues for the first two principal components (F1 and F2) are presented in Table 1. The first two PCAs extracted from the components amounted to 77.06% of the total data variance for 50 μ mol m −2 s −1 , 81.74% for 100 μ mol m −2 s −1 , and 90.10% for 100 μ mol m −2 s −1 PFDs. In PCA biplots ( Figure 2C). The PCA biplots generally showed distinct effects of B and G to R and FR regardless of PFD. The PCA factor loadings, scores, and eigenvalues for the first two principal components (F1 and F2) are presented in Table 1. The first two PCAs extracted from the components amounted to 77.06% of the total data variance for 50 µmol m −2 s −1 , 81.74% for 100 µmol m −2 s −1 , and 90.10% for 100 µmol m −2 s −1 PFDs. In PCA biplots (Figure 2A-C), two vectors with an angle <90 • show a positive correlation, and two vectors with an angle >90 • have a negative correlation. At 50 µmol m −2 s −1 , moderate significant positive correlations between 1 DAI, 2 DAI, and 4 DAI, and weak significant positive correlation between 2 DAI and 3 DAI (Table S1). At 100 µmol m −2 s −1 PFD, a significantly strong correlation between 1 DAI and 2 DAI was determined, and a significant moderate correlation between 2 DAI and 4 DAI (Table S2). At 200 µmol m −2 s −1 PFD, a significant very strong correlation between 2 DAI and 3 DAI and 4 DAI, a significantly strong correlation between 3 DAI and 4 DAI, and 1 DAI with 3 DAI, as well as a significant moderate correlation between 1 DAI and 2 DAI, was observed (Table S3).
DAI and 3 DAI (Table S1). At 100 μ mol m s PFD, a significantly strong correlation between 1 DAI and 2 DAI was determined, and a significant moderate correlation between 2 DAI and 4 DAI (Table S2). At 200 μ mol m −2 s −1 PFD, a significant very strong correlation between 2 DAI and 3 DAI and 4 DAI, a significantly strong correlation between 3 DAI and 4 DAI, and 1 DAI with 3 DAI, as well as a significant moderate correlation between 1 DAI and 2 DAI, was observed (Table S3).   Table 1. Factor loadings, eigenvalues, variability (%), cumulative variability (%), and scores for the 50 (A), 100 (B) and 200 (C) µmol m −2 s −1 photon flux density for C. acutatum mycelium growth measurements under five different LED light wavelengths on first-four days after inoculation. The PCA Biplot A, B, C indicates treatments of 50, 100, and 200 µmol m −2 s −1 photon flux density, respectively. DAI-days after inoculation; B-blue (peak = 450 nm); G-green (peak = 530 nm); R-red (peak = 660 nm); FR-far red (peak = 735 nm); W-white (5700 K). To evaluate the associations between DAI's and lighting treatments on C. acutatum mycelium growth, the PCA biplots were analyzed according to F1 and F2 factor loadings and scores (Table 1; Figure 2A-C). At 50 µmol m −2 s −1 PFD, the associations between 1 DAI and 4 DAI to R and FR lighting treatments were found. In addition, 2 DAI and 3 DAI were associated with W lighting. None of the DAI's was associated with B and G treatments (Figure 2A). At 100 µmol m −2 s −1 PFD, 1 DAI was associated with G, 2 DAI and 4 DAI to B, and 3 DAI to R, FR, and W lighting treatments ( Figure 2B). At 200 µmol m −2 s −1 PFD, 1 DAI was associated with FR and W, and 2 DAI, 3 DAI, and 4 DAI to R treatment. It was found that B and G lighting treatments were not associated with C. acutatum mycelium growth at all investigated DAIs at 200 µmol m −2 s −1 PFD ( Figure 2C).

PCA Biplot
The principal component analysis demonstrated distinct effects of lighting treatments at different PFD levels (50, 100 and 200 µmol m −2 s −1 ) on C. acutatum mycelium growth at all investigated days after infection (1 to 4 DAI). However, similar trends were observed between associations of 1 DAI and 4 DAI to R and FR lighting at 50 and 200 µmol m −2 s −1 PFDs. Contrary to this, at 100 µmol m −2 s −1 PFD, such lighting treatments were associated with 3 DAI.
The color of C. acutatum upper mycelium under different wavelengths varied irrespectively to PFD (Figure 3). The C. acutatum colonies could be grouped into three types based on the basis observed in color pigments. The same color tendency was under all three evaluated PFD. The color ranged from white to orange. The C. acutatum mycelium color under G, R and FR was light orange, and under B and W-orange color at 5 DAI after incubation under different wavelengths. Overall, C. acutatum colonies' color varied among different LED light wavelengths. To determine the recovery of the C. acutatum after different LEDs and PFD influence, the re-isolation was after 5 DAI (Table 4). C. acutatum colonies, after reisolation, visually looked similar to the original isolates. Results are presented as mean ± SD (n = 4) (p < 0.05). According to Duncan's multiple range test, the means of different letters are significantly different at the p < 0.05 level. LED light wavelengths: B-blue (peak = 450 nm); G-green (peak = 530 nm); R-red (peak = 660 nm); FR-far red (peak = 735 nm); W-white (5700 K).
However, the data revealed that recovery was slower at 50 μ mol m −2 s −1 PFD under B than other wavelengths. Additionally, under R recovered faster than under other To determine the recovery of the C. acutatum after different LEDs and PFD influence, the re-isolation was after 5 DAI (Table 4). C. acutatum colonies, after re-isolation, visually looked similar to the original isolates. Results are presented as mean ± SD (n = 4) (p < 0.05). According to Duncan's multiple range test, the means of different letters are significantly different at the p < 0.05 level. LED light wavelengths: B-blue (peak = 450 nm); G-green (peak = 530 nm); R-red (peak = 660 nm); FR-far red (peak = 735 nm); W-white (5700 K).
However, the data revealed that recovery was slower at 50 µmol m −2 s −1 PFD under B than other wavelengths. Additionally, under R recovered faster than under other LEDs. However, at 100 µmol m −2 s −1 PFD, the slowest recovery was observed under W and

Discussion
The LEDs could be an innovative tool for environmentally friendly pathogen control in controlled environmental conditions. The LEDs allow a selection of a specific light spectrum and PFD to prevent horticultural crops damaged by pathogens. However, literature research showed a lack of studies on the light-mediated effect on specific wavelength and PFD, but not as in our study [29,30,[33][34][35]. Fungi realize red, green, near-ultraviolet, blue, and far-red with 11 photoreceptors. Mostly all fungi have two types of photoreceptors, while some even have three. In addition, light for fungi may give an orientation for reproduction and growth [27,28].
Fungi use light combined with the circadian clock to adapt to stress and reproductive structures productions at precise time and place [36]. This study observed that C. acutatum acted differently under various LEDs and PFD. Different LEDs and PFD could suppress or stimulate plant pathogens as fungi respond to light [8,9,14,27,28,35]. Therefore, the LEDs and PFD inhibitory effect could be related to the pathogen's ability to infect plant tissues and spread. As light influences plant growth and development for all crops, selecting specific spectra eliminates cropping season in closed environment conditions and influences plants' grown parameters and nutritional quality [22][23][24][25]. Therefore, a knowledge of LEDs and PDF effect on C. acutatum is essential for combined plants cultivation in closed environmental conditions. The present study focused on the various LEDs and PFD effects on C. acutatum in vitro.
Recently, several reports had a light effect on the development, growth of fungi, and inhibition. Most studies agree that light influences various fungal pathogens, not many on C. acutatum. For example, it's reported that light quality influences rose Podosphaera pannosa development and growth [35]. The continuous blue light (40 µmol m −2 s −1 ) or dark-light regime reduced the mycelium growth of Penicillium italicum and P. citri [30]. In addition, B. cinerea illumination by purple light (400-410 nm) and blue (450-460 nm at 12 h photoperiod inhibited mycelium growth [33]. Besides this, C. capsici lowest growth was under 24 h dark photoperiod [37]. In addition, C. gloeosporioides irradiation by continuous 25 mW/cm 2 of 405 nm light for 40 min causes morphological changes in its colony [38]. These findings indicate that various fungi mycelium growth was affected under different light. Our results showed the highest inhibition of C. acutatum after 4 DAI at 50 µmol m −2 s −1 PFD was under G, at 100 µmol m −2 s −1 PFD-under R, and at 200 µmol m −2 s −1 PFD-under B. On the other hand, the presence of light could influence spore production. C. gloeosporioides sporulated intensely under 16 h of light [39]. In addition, Podosphaera pannosa conidia germination was reduced by blue (420 to 520 nm, peak 465 nm) light [34]. Additionally, blue (410-540 nm) light inhibits spore production of P. digitatum [30]. C. acutatum colony morphology differed under various PFD, and a 6 h photoperiod of blue (458 nm) LED light influenced its conidial germination and germ tube growth [36]. According to these results, light inhibits or reduces spore production. These findings could serve to pathogen ability to spread. Our results revealed that PFD and LED light influenced conidia size. Furthermore, as in control environment conditions, supplemental light is essential. Therefore, as light influences fungal pathogens, the selection of specific LEDs and PFD could help suppress and control strawberry fungal pathogen C. acutatum. LEDs in plant protection leads to optimal and appropriate technology usage. In addition, LEDs illumination can be combined with other disease control methods. The findings of this study will serve for future research.

Materials and Methods
The experiments were carried out at the Lithuanian Research Centre for Agriculture and Forestry (LAMMC IH), Institute of Horticulture, Laboratory of Plant Physiology under closed, controlled environment conditions in 2018-2019.

Fungal Isolate and Its Cultivation
Single spore C. acutatum F-05-I isolate was obtained from LAMMC IH Laboratory of plant protection isolate collection. The F-05-I origin was from the rotten strawberry fruit. The isolate was identified by PCR as C. acutatum by Xie et al. [40]. For experiments, isolates were cultivated in Petri plates containing potato dextrose agar (PDA) (Liofilchem, Roseto degli Abruzzi, Italy) at 25 ± 2 • C for 7 DAI. The isolates mycelial plugs (7 mm diameter) mycelium were placed side-down in the center of the new Petri with PDA. The morphological and phenotypic characteristics of C. acutatum were evaluated under different LEDs and PFD. The closed, controlled environment conditions were 23 ± 2 • C temperature and four hours (h) photoperiod. The control plates were incubated in complete darkness. There were four replicates per treatment.

Light Treatments and Intensity
For experiments, five different LEDs and three PFD treatments were selected. The light source was six LEDs wavelengths, as follows: B-blue (peak = 450 nm), G-green (peak = 530 nm), R-red (peak = 660 nm), FR-far red (peak = 735 nm), and W-white (5700 K) (Heliospectra RX30, Gothenburg, Sweden). The PFD was 50, 100 and 200 µmol m −2 s −1 . The intensity of light was measured by photometer-radiometer RF-100 (Sonopan, Bialystok, Poland). The distance from the fungal sample to the light source was 40 cm.

Phenotypic and Morphological Characteristics Evaluation
C. acutatum was evaluated for phenotypic (conidia presence and size, mycelium growth rate) and morphological (mycelium appearance) characteristics. The mycelium growth rate was evaluated by measuring isolates diameter daily (mm day −1 ) and calculated as an average length and width increase per day for five days after inoculation (1-5 DAI).
The mycelium growth rate was used for mycelial growth inhibition (MGI) calculation [41].
where C-C. acutatum mycelium growth rate diameter in control, mm; T-C. acutatum mycelium growth rate diameter in treatment, mm. Mycelial growth curve (AUMGC) was calculated by the formula [42] AUMGC = ∑ ((y i + y i + 1)/2 × dti)/n; where y i = diameter of mycelium (mean) in the four observation, mm, dti = interval between evaluations, n = evaluation period. The appearance of mycelium, presence of conidia, and size (width and length) were evaluated after 5 DAI. A conidia size (width and length, µm) and presence were evaluated in the plate's margin with a Nikon Eclipse 80i microscope of 40× magnification after 5 DAI. The color of mycelium upper side at white and background evaluated 5 DAI. The mycelium color types were as follows: (1) white, (2) light orange, (3) orange. Three replications were maintained for each light and PFD treatment.
The re-isolation after 5 DAI was to evaluate the recovery of C. acutatum after treatment. The re-isolated growth rate (mm) measured after 48 h.

Statistical Analysis and Principal Component Analysis
The experimental data were analyzed with the software ANOVA from the program SAS Enterprise Guide, version 7.1 (SAS Inc., Cary, NC, USA). The standard error (SE) marked in the figures as an error bar was estimated for the growth rates of the isolates. Duncan's Multiple Range Test used to determine differences among treatments. Conidia size and re-isolation data expressed as mean ± standard deviation.
The principal component analysis (PCA) was performed using Addinsoft XLSTAT 2019.1 XLSTAT statistical and data analysis solution (Long Island, NY, USA). The results presented in PCA Biplots indicate distinct effects of lighting treatments (B, G, R, FR, W) on C. acutatum mycelium growth at 1, 2, 3 and 4 days after infection (1 DAI, 2 DAI, 3 DAI, and 4 DAI, respectively) (based on Pearson's correlation matrix).

Conclusions
Our results allowed exploring original research data techniques and leads to green plant protection solutions. Additionally, it raised new scientific questions for further research to develop environmentally safe plant protection methods of C. acutatum management in closed environmental conditions. The highest inhibition of C. acutatum achieved at 50 µmol m −2 s −1 PFD under G, at 100 µmol m −2 s −1 PFD-R, and 200 µmol m −2 s −1 PFD under B after 4 DAI. The lowest conidia width was under G and length under R. The C. acutatum mycelium color varied, and under G, R, and FR was light orange, and under B and W-orange color under different wavelengths at 5 DAI. The slowest recovery was at 50 µmol m −2 s −1 PFD under B, but at 100 µmol m −2 s −1 PFD-under W, and at 200 µmol m −2 s −1 PFD-under B. In conclusion, different PFD and LEDs influence C. acutatum growth characteristics. Furthermore, LEDs and PFD effects on C. acutatum growth characteristics are valuable for strawberries and other crops exposed to this pathogen. However, this research is in the initial stage, and further research is needed to develop innovative plant protection techniques for strawberry C. acutatum control.